Ni nta column

Manufactured by Bio-Rad

Sourced in United States

The Ni-NTA column is a chromatography column used for the purification of proteins. It contains a resin that has nickel ions (Ni2+) immobilized on its surface, which can bind to proteins with a histidine tag (His-tag) on their structure. This allows for the selective capture and separation of the target protein from complex mixtures.

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Lab products found in correlation

Trizol reagent, by Thermo Fisher Scientific (2 mentions) Nanodrop uv vis spectrophotometer, by Thermo Fisher Scientific (1 mentions) Ab5131, by Merck Group (1 mentions) Ab9050, by Abcam (1 mentions) α flag, by Merck Group (1 mentions) Kanamycin, by Merck Group (1 mentions) Anti cdk6 antibody, by Cell Signaling Technology (1 mentions) Ni nta bead, by Qiagen (1 mentions) Isopropyl β d 1 thiogalactopyranoside (iptg), by Merck Group (1 mentions) Corresponding restriction enzymes, by Thermo Fisher Scientific (1 mentions) Hitrap desalting column, by Cytiva (1 mentions) Q5 polymerase, by New England Biolabs (1 mentions) Mmlv rt kit, by Evrogen (1 mentions) Ultrasonicator, by Sonics (1 mentions) Enterokinase, by GE Healthcare (1 mentions) Pd 10 desalting column, by GE Healthcare (1 mentions) Ni nta column, by GE Healthcare (1 mentions)

8 protocols using ni nta column

Protein Purification and Western Blot Analysis

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Luria–Bertani
broth (Merck, Darmstadt,
Germany), kanamycin, IPTG (Sigma, Saint Louis, MO, USA), Ni-NTA column
(Biorad), and Ni-NTA beads (Qiagen QIA express) were purchased. The
Ni-NTA column purchased from Biorad (Qiagen QIA express). Anti-Cdk6
antibody (Cat#mAb #13331) is from Cell Signaling Technology, Danvers,
USA, and anti-β-Actin monoclonal antibody (Cat#A2228) is from
Millipore Sigma, Darmstadt, Germany. Cancer cells were procured from
National Centre for Cell Sciences (NCCS), Pune, India. Primary antibody
goat anti-rabbit IgG (31635) secondary antibodies and dihydroethidium
were taken from Invitrogen; Dulbecco minimal essential medium (DMEM),
RPMI-1640, and fetal bovine serum (FBS) were purchased from Gibco
life sciences. All reagents used were molecular biology grades.

Yousuf M., Khan P., Shamsi A., Shahbaaz M., Hasan G.M., Haque Q.M., Christoffels A., Islam A, & Hassan M.I. (2020). Inhibiting CDK6 Activity by Quercetin Is an Attractive Strategy for Cancer Therapy. ACS Omega, 5(42), 27480-27491.

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Protein Purification via Ultrafiltration and Affinity Chromatography

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The harvested media containing the secreted proteins were concentrated 10- to 20-fold on a Prep/Scale Spiral Wound 10-KDa MWCO Ultrafiltration module (Millipore, cat. # SK1P003W4). Recombinant proteins were purified from the concentrated media by immobilized-metal affinity chromatography on Ni-NTA columns (Bio-Rad, cat. # 156-0135). Purified proteins were quantified using absorbance at A280 nm (NanoDrop UV-Vis spectrophotometer; Thermo Scientific) and confirmed by Coomassie blue staining after SDS–PAGE.

Michael I.P., Westenskow P.D., Hacibekiroglu S., Greenwald A.C., Ballios B.G., Kurihara T., Li Z., Warren C.M., Zhang P., Aguilar E., Donaldson L., Marchetti V., Baba T., Hussein S.M., Sung H.K., Iruela-Arispe M.L., Rini J.M., van der Kooy D., Friedlander M, & Nagy A. (2014). Local acting Sticky-trap inhibits vascular endothelial growth factor dependent pathological angiogenesis in the eye. EMBO Molecular Medicine, 6(5), 604-623.

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Purification and Characterization of dBigH1

Cited 1 time

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αdBigH1 antibodies are described in (6 (link)). αdH1 antibodies were a gift from Dr J. Kadonaga and are described in (16 (link)). αRpb3 antibodies were a gift from Dr J. Zeitlinger and are described in (17 (link)). The rest of antibodies used in these experiments were commercially available: αPol IIo^ser5 (Abcam, ab5131), αH3Kac (Millipore 06-599), αH3K36me3 (Cell Signaling, 9715), αH3 (Abcam, ab9050), αH4 (Abcam, ab10158) and αFLAG (Sigma F3165).
Recombinant His-tagged dBigH1 constructs were expressed in Escherichia coli BL21-LysS cells using pET30b(+) expression vectors (Novagen), and purified using Ni-NTA columns (BioRad) by conventional methods.

Climent-Cantó P., Carbonell A., Tatarski M., Reina O., Bujosa P., Font-Mateu J., Bernués J., Beato M, & Azorín F. (2020). The embryonic linker histone dBigH1 alters the functional state of active chromatin. Nucleic Acids Research, 48(8), 4147-4160.

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Recombinant Protein Expression and Purification

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The ORFs of desired genes were amplified from cDNAs of the interest, cloned in pET28a(+) and transformed into E. coli BL21-CodonPlus (DE3)-RIPL cells. Protein expression was induced with 0.5 mM Isopropyl β-D-thiogalactoside (IPTG) for 6 h at 23°C. Bacterial pellet was lysed in buffer [500 mM NaCl, 50 mM NaPO₄ (pH 8.0), 10 mM Imidazole, 1mM β-Mercaptoethanol, 1 mg/mL lysozyme and 1mM Phenylmethylsulfonyl fluoride (PMSF)] by incubating 30 min on ice, followed by sonication (10 cycle, 70% amplitute). The cell lysates were precipitated followed by 0.45 μm filtration. The cleared lysate was incubated with Ni-NTA resin for 30 min at 4°C. The His-tagged fusion proteins were purified using a Ni-NTA column (Bio-Rad, USA). Proteins were eluted in elution buffer [500 mM NaCl, 50 mM NaPO₄ (pH 8.0), 10% glycerol and 200 mM imidazole]. The quality and quantity of eluted protein were checked by SDS/PAGE and Bradford assay, respectively. The proteins were dialyzed in compatible buffers, as per experiment, followed by concentration.

Sinha M., Shree A., Singh K., Kumar K., Singh S.K., Kumar V, & Verma P.K. (2021). Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogen Ascochyta rabiei. PLoS Genetics, 17(5), e1009137.

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TBEV Antigen Production for ELISA

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Recombinant domains of the protein E (sE and dIII) of the Siberian TBEV subtype strain Sukhar were used as antigens in the ELISA test system [33 (link)].
TBEV (Sukhar strain) RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Waltham, MA, USA). The cDNA was obtained using an MMLV RT kit (Evrogen, Moscow, Russia) following the manufacturer’s instructions. Target genes were cloned with Q5 polymerase (New England Biolabs, Ipswich, MA, USA), digested with corresponding restriction enzymes (Thermo Fisher Scientific, Waltham, MA, USA), and ligated into pQE vectors (Table 2). E. coli cells (strain JM109) were transiently transformed with the obtained plasmids. His-tagged recombinant viral antigens were purified on a Ni–NTA column (Bio-Rad, Hercules, CA, USA). After purification, the buffer was exchanged for a storage 1× PBS buffer (FSASI Chumakov FSC R&D IBP RAS, Moscow, Russia) using HiTrap desalting columns (Cytiva, Marlborough, MA, USA).

Baryshnikova V., Turchenko Y., Tuchynskaya K., Belyaletdinova I., Butenko A., Dereventsova A., Ignatiev G., Kholodilov I., Larichev V., Lyapeykova E., Rogova A., Shakaryan A., Shishova A., Gmyl A, & Karganova G. (2023). Recombinant TBEV Protein E of the Siberian Subtype Is a Candidate Antigen in the ELISA Test System for Differential Diagnosis. Diagnostics, 13(20), 3277.

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Recombinant Nanosensor Protein Purification

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Escherichia coli BL21 (DE3) cells carrying the nanosensor construct were grown on Luria–Bertani (LB) medium containing ampicillin at 20°C for 24 h. Induction of the nanosensor protein was carried out by adding 0.5 mM Isopropyl β-D-1-thiogalactopyranoside, Isopropyl β-Dthiogalactoside (IPTG) (Fermentas, Germany). After induction, cells were kept for 48 h in the dark at 16°C with continuous shaking. The cells were then harvested and centrifuged at 6,500 × g at 4°C for 30 min, for the isolation of the nanosensor protein. The cell pellet was resuspended in chilled Tris-HCl buffer (20 mM, pH 7.2). An ultrasonicator (Sonics, USA) was used for bacterial cells lysis, followed by centrifugation and filtration of the lysate, to remove cell debris. The isolated protein fraction was collected and applied to nickel-aminotriacetic acid (Ni-NTA) column (BioRad, CA, USA) for His-tag-mediated purification of the nanosensor protein. After washing of the column, elution of the sensor protein was carried out by applying elution buffer (20 mMTris-Cl, pH 8.0, 250 mM imidazole). The eluted protein was incubated at 4°C for 12 h, for proper folding.

Naz R., Okla M.K., Fatima U., Mohsin M., Soufan W.H., Alaraidh I.A., Abdel-Maksoud M.A, & Ahmad A. (2021). Designing and Development of FRET-Based Nanosensor for Real Time Analysis of N-Acetyl-5-Neuraminic Acid in Living Cells. Frontiers in Nutrition, 8, 621273.

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Recombinant Protein Purification Protocol

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Respective cDNAs were cloned in pET28a (+) and transformed into E. coli BL21-CodonPlus (DE3)-RIPL cells. The protein expression was induced with 0.5 mM Isopropyl β-Dthiogalactoside (IPTG) for 6 h at 23ºC. Bacterial pellet was lysed in buffer [500 mMNaCl, 50 mM NaPO 4 (pH 8.0), 10 mM Imidazole, 1mM β-Mercaptoethanol, 1 mg/ml lysozyme and 1mM Phenylmethylsulfonyl fluoride (PMSF)] by incubating 30 min on ice, followed by sonication.
The cell lysates were precipitated followed by 0.45 µm filtration. The cleared lysate was incubated with Ni-NTA resin for 30 min at 4ºC. The His-tagged fusion protein was purified using a Ni-NTA column (Bio-Rad, USA). Protein was eluted in elution buffer [500 mMNaCl, 50 mM NaPO 4 (pH 8.0), 10% glycerol and 200 mM imidazole]. The quality and quantity of eluted protein were checked by SDS/PAGE and Bradford assay, respectively. The proteins were dialyzed in respective compatible buffers followed by concentration.

Sinha M., Shree A., Singh K., Kumar K., Kumar V., & Verma P.K. (2020). Modulation of fungal virulence through CRZ1 regulated F-BAR-dependent actin remodeling and endocytosis in chickpea infecting phytopathogenAscochyta rabiei.

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Purification of Mouse α-Syntrophin PDZ Domain

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The pET32a plasmid containing the mouse α-syntrophin PDZ domain sequence as a thioredoxin (TRX) fusion protein, with a centrally located His-tag, was expressed and purified as in (Seedorff et al. 2010) . Briefly, the plasmid was transformed into BL21 (DE3) cells, which were grown to OD 0.8 in LB at 37°C, before induction with 0.1 mM IPTG and transferred to 30°C for 3.5 h. The culture was centrifuged and the cell pellet lysed by repetitive freezethawing, before further centrifugation. The clarified lysate was applied to a Ni-NTA column (BioRad) washed, and eluted with increasing concentrations of imidazole. Eluted fractions were combined and buffer exchanged using a PD10 desalting column (GE Healthcare) to remove imidazole, before treatment with enterokinase (Genscript, 50 units/10mg purified protein) at 20°C for 20 h. The His-tagged TRX domain and enterokinase were removed by reapplication to the Ni-NTA column. Purified PDZ domain was concentrated and lyophilized for storage. Immediately prior to the electrochemical experiments, the domain was resuspended and exchanged into 50 mM HEPES (pH=8.5) and 150 mM NaCl, at a final concentration of 230 μM, based on an extinction coefficient of 2980 M -1 cm -1 (λ=280 nm).

Horsley J.R., Yu J., Wegener K.L., Hoppmann C., Rück-Braun K, & Abell A.D. (2018). Photoswitchable peptide-based 'on-off' biosensor for electrochemical detection and control of protein-protein interactions. Biosensors & bioelectronics, 118.

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